208 research outputs found
Polarity of T Cell Shape, Motility, and Sensitivity to Antigen
AbstractT cell activation requires contact with APCs. We used optical techniques to demonstrate T cell polarity on the basis of shape, motility, and localized sensitivity to antigen. An intracellular Ca2+ clamp showed that T cell shape and motility are extremely sensitive to changes in [Ca2+]i (Kd = 200 nM), with immobilization and rounding occurring via a calcineurin-independent pathway. Ca2+-dependent immobilization prolonged T cell contact with the antigen-presenting B cell; buffering the [Ca2+]i signal prevented the formation of stable cell pairs. Optical tweezers revealed spatial T cell sensitivity to antigen by controlling placement on the T cell surface of either B cells or α-CD3 MAb-coated beads. T cells were 4-fold more sensitive to contact made at the leading edge of the T cell compared with the tail. We conclude that motile T cells are polarized antigen sensors that respond physically to [Ca2+]i signals to stabilize their interaction with APCs
Brain Endothelial Erythrophagocytosis and Hemoglobin Transmigration Across Brain Endothelium: Implications for Pathogenesis of Cerebral Microbleeds
Peripheral endothelial cells are capable of erythrophagocytosis, but data on brain endothelial erythrophagocytosis are limited. We studied the relationship between brain endothelial erythrophagocytosis and cerebral microhemorrhage, the pathological substrate of MRI-demonstrable cerebral microbleeds. To demonstrate the erythrophagocytic capability of the brain endothelium, we studied the interactions between brain endothelial cells and red blood cells exposed to oxidative stress in vitro, and developed a new in vitro cerebral microbleeds model to study the subsequent passage of hemoglobin across the brain endothelial monolayer. Using multiple approaches, our results show marked brain endothelial erythrophagocytosis of red blood cells exposed to oxidative stress compared with control red blood cells in vitro. This brain endothelial erythrophagocytosis was accompanied by passage of hemoglobin across the brain endothelial monolayer with unaltered monolayer integrity. In vivo and confocal fluorescence microscopy studies confirmed the extravasation of RBC exposed to oxidative stress across brain endothelium. These findings, demonstrating erythrophagocytosis mediated by the brain endothelial monolayer and the subsequent passage of iron-rich hemoglobin in vitro and RBC in vivo, may have implications for elucidating mechanisms involved in the development of cerebral microbleeds that are not dependent on disruption of the microvasculature
Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells
Damage recognition by repair/checkpoint factors is the critical first step of the DNA damage response. DNA double strand breaks (DSBs) activate checkpoint signaling and are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways. However, in vivo kinetics of the individual factor responses and the mechanism of pathway choice are not well understood. We report cell cycle and time course analyses of checkpoint activation by ataxia-telangiectasia mutated and damage site recruitment of the repair factors in response to laser-induced DSBs. We found that MRN acts as a DNA damage marker, continuously localizing at unrepaired damage sites. Damage recognition by NHEJ factors precedes that of HR factors. HR factor recruitment is not influenced by NHEJ factor assembly and occurs throughout interphase. Damage site retention of NHEJ factors is transient, whereas HR factors persist at unrepaired lesions, revealing unique roles of the two pathways in mammalian cells
Rat reproductive performance following photodynamic therapy with topically administered Photofrin
A rat animal model was used for comparing the photodynamic efficacy of two formulations of topically administered Photofrin in the uterus: 0.7 mg/kg Photofrin and 0.7 mg/kg Photofrin + 4% Azone, a penetrationenhancing agent. Uterine structure and reproductive performance were evaluated following illumination with 80 J/cm2 of 630 nm light. Fluorescence microscopy was employed to determine drug localization in frozen uterine sections at various times after drug administration. Functionality studies demonstrated a significant reduction in the number of implantations per treated uterine horn compared to controls. The mean number of implantations decreased systematically on increasing the interval between Photofrin administration and light application. At 72 h, 0.88 ± 0.52 gestational sacs per rat were recorded with Photofrin therapy, compared with 8.1 ± 1.12 (P = 0.01) on the untreated side, indicating nearly complete loss of reproductive capability. Similar results were achieved after only 3 h treatment with Photofrin + Azone (0.38 ± 0.26 sacs per rat versus 7.5 ± 1.07 on the untreated side; P = 0.01). This indicates that the effect of Photofrin can be enhanced either by extending the drug incubation period from 3 to 72 h or by adding the penetration-enhancing drug Azone. Fluorescence pharmacokinetic studies suggest that both forms of topically administered Photofrin are diffusely distributed throughout the endometrium at virtually the same rate. However, Azone may enhance the selectivity of photodynamic therapy by facilitating drug targeting to critical endometrial structure
A Murine Model of Inflammation-Induced Cerebral Microbleeds
Background: Cerebral microhemorrhages (CMH) are tiny deposits of blood degradation products in the brain and are pathological substrates of cerebral microbleeds. The existing CMH animal models are β-amyloid-, hypoxic brain injury-, or hypertension-induced. Recent evidence shows that CMH develop independently of hypoxic brain injury, hypertension, or amyloid deposition and CMH are associated with normal aging, sepsis, and neurodegenerative conditions. One common factor among the above pathologies is inflammation, and recent clinical studies show a link between systemic inflammation and CMH. Hence, we hypothesize that inflammation induces CMH development and thus, lipopolysaccharide (LPS)-induced CMH may be an appropriate model to study cerebral microbleeds.
Methods: Adult C57BL/6 mice were injected with LPS (3 or 1 mg/kg, i.p.) or saline at 0, 6, and 24 h. At 2 or 7 days after the first injection, brains were harvested. Hematoxylin and eosin (H&E) and Prussian blue (PB) were used to stain fresh (acute) hemorrhages and hemosiderin (sub-acute) hemorrhages, respectively. Brain tissue ICAM-1, IgG, Iba1, and GFAP immunohistochemistry were used to examine endothelium activation, blood-brain barrier (BBB) disruption, and neuroinflammation. MRI and fluorescence microscopy were used to further confirm CMH development in this model.
Results: LPS-treated mice developed H&E-positive (at 2 days) and PB-positive (at 7 days) CMH. No surface and negligible H&E-positive CMH were observed in saline-treated mice (n = 12). LPS (3 mg/kg; n = 10) produced significantly higher number, size, and area of H&E-positive CMH at 2 days. LPS (1 mg/kg; n = 9) produced robust development of PB-positive CMH at 7 days, with significantly higher number and area compared with saline (n = 9)-treated mice. CMH showed the highest distribution in the cerebellum followed by the sub-cortex and cortex. LPS-induced CMH were predominantly adjacent to cerebral capillaries, and CMH load was associated with indices of brain endothelium activation, BBB disruption, and neuroinflammation. Fluorescence microscopy confirmed the extravasation of red blood cells into the brain parenchyma, and MRI demonstrated the presence of cerebral microbleeds.
Conclusions: LPS produced rapid and robust development of H&E-positive (at 2 days) and PB-positive (at 7 days) CMH. The ease of development of both H&E- and PB-positive CMH makes the LPS-induced mouse model suitable to study inflammation-induced CMH
Smoking cessation and depression after acute coronary syndrome.
Smoking and depression are risk factors for acute coronary syndrome (ACS) that often co-exist. We investigated the evolution of depression according to smoking cessation one-year after ACS. Data from 1822 ACS patients of the Swiss multicenter SPUM-ACS cohort study were analyzed over a one-year follow-up. Participants were classified in three groups based on smoking status one-year post-ACS - continuous smokers, smokers who quit within the year, and non-smokers. Depression status at baseline and one-year was assessed with the Center for Epidemiologic Studies Depression scale (CES-D) and antidepressant drug use. A CES-D score ≥ 16 defined depression. A multivariate-adjusted logistic regression model was used to calculate odds ratios (OR) between groups. The study sample mean age was 62.4 years and females represented 20.8%. At baseline, 22.6% were depressed, 40.9% were smokers, and 47.5% of these quit smoking over the year post-ACS. In comparison to depressed continuous smokers, depressed smokers who quit had an adjusted OR 2.59 (95% confidence interval (CI) 1.27-5.25) of going below a CES-D score of 16 or not using antidepressants. New depression at one-year was found in 24.4% of non-depressed smokers who quit, and in 27.1% of non-depressed continuous smokers, with an adjusted OR 0.85 (95% CI 0.55-1.29) of moving to a CES-D score of ≥16 or using antidepressants. In conclusion, smokers with depression at time of ACS who quit smoking improved their depression more frequently compared to continuous smokers. The incidence of new depression among smokers who quit after ACS was similar compared to continuous smokers
ATR Mutations Promote the Growth of Melanoma Tumors by Modulating the Immune Microenvironment.
Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet somehow suppress the immune response to facilitate continued growth. In this study, we identify a subset of human melanomas that have loss-of-function mutations in ATR, a kinase that recognizes and repairs UV-induced DNA damage and is required for cellular proliferation. ATR mutant tumors exhibit both the accumulation of multiple mutations and the altered expression of inflammatory genes, resulting in decreased T cell recruitment and increased recruitment of macrophages known to spur tumor invasion. Taken together, these studies identify a mechanism by which melanoma cells modulate the immune microenvironment to promote continued growth
Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media
Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy
- …